LID scientists are collaborating with scientists from MedImmune under a CRADA to generate candidate vaccines against avian influenza viruses of each subtype, including H5N1 viruses that have caused human infections since 2003. The vaccines were generated using plasmid based reverse genetics and each contains the hemagglutinin and neuraminidase genes from an avian influenza virus and six internal gene segments from the AA ca virus. Highly pathogenic avian influenza is a disease of poultry that is caused by H5 or H7 avian influenza viruses and is associated with up to 100% mortality. H7 subtype viruses have significant pandemic potential. Influenza A H7 subtype viruses from both Eurasian and North American lineages have resulted in more than 100 cases of human infection since 2002 in the Netherlands, Italy, Canada, the United Kingdom, and the United States. A live attenuated H7N7 candidate vaccine virus was generated by reverse genetics using the modified hemagglutinin (HA) and neuraminidase (NA) genes of HP A/Netherlands/219/03 (NL/03) (H7N7) wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (AA ca) (H2N2) virus. The reassortant H7N7 NL/03 ca vaccine virus was temperature sensitive and attenuated in mice, ferrets, and African green monkeys (AGMs). Intranasal administration of a single dose of the H7N7 NL/03 ca vaccine virus fully protected mice from lethal challenge with homologous and heterologous H7 viruses from Eurasian and North American lineages. Two doses of the H7N7 NL/03 ca vaccine induced neutralizing antibodies in the serum and provided complete protection from pulmonary replication of homologous and heterologous wild-type H7 challenge viruses in mice and ferrets. One dose of the H7N7 NL/03 ca vaccine elicited an antibody response in one of three AGMs that was completely protected from pulmonary replication of the homologous wild-type H7 challenge virus. The contribution of CD8+ and/or CD4+ T cells to the vaccine-induced protection in mice was evaluated by T cell depletion;T lymphocytes were not essential for the vaccine-induced protection from lethal challenge with H7 wt viruses. Additionally, passively transferred neutralizing antibody induced by H7N7 NL/03 ca virus protected mice from lethality following challenge with H7 wt viruses. The safety, immunogenicity, and efficacy of H7N7 NL/03 ca vaccine virus in mice, ferrets and AGMs support the evaluation of this vaccine virus in Phase 1 clinical trials. While LAIVs have been shown to be efficacious and have been licensed for human use, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) have to be updated for optimal protective efficacy. Little is known about the effect of different HA and NA proteins on the immunogenicity of LAIVs developed using the same backbone. A panel of LAIVs that share the internal protein genes, with unique HA and NA gene segments from different influenza subtypes were rescued by reverse genetics and a comparative study of immune responses induced by these vaccines were conducted in mice. The results suggest that the magnitude of lung immunity, including pulmonary IgA antibody and memory CD8+ T lymphocytes induced by the vaccines depends on the replication efficiency of the LAIVs, as well as the induction of cytokines/chemokines in the lungs. However, these factors are not important in determining systemic immunity such as serum antibody titers and memory CD8+ T cells in the spleen. A qualitative analysis of immune responses induced by a single dose of an H5N1 LAIV revealed that the vaccine induced robust systemic and mucosal immunity in mice. In addition, antibodies and memory lymphocytes established in the lungs following vaccination were required for protection against lethal challenge with homologous and heterologous H5N1 viruses. Our results highlight the different requirements for inducing systemic and lung immunity that can be explored for the development of pulmonary immunity for protection against respiratory pathogens. Several H5N1 LAIV vaccines possessing a modified HA and neuraminidase (NA) of an H5N1 virus and the six internal protein gene segments of the A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) master donor virus were previously generated and evaluated for their immunogenicity and efficacy in mice and ferrets. A single dose of A/Vietnam/1203/2004 (VN04 ca) LAIV elicited very low levels of serum neutralizing antibodies against homologous and heterologous wild-type (wt) H5N1 viruses four weeks after administration to mice and ferrets. In contrast, a single dose of A/Hong Kong/213/2003 (H5N1) (HK03 ca) LAIV was more immunogenic. A specific amino acid residue at position 227 in the HK03 HA has been reported to be responsible for the greater immunogenicity of HK03. VN04 and HK03 also differ in their receptor binding specificity. The VN04 HA mainly recognizes &#945;2,3SAL while the HK03 HA recognizes both &#945;2,3SAL and &#945;2,6SAL. Sequence alignment of the two H5 HA proteins revealed nine amino acid (aa) differences in their HA1 region. We evaluated the impact of these amino acid differences on H5N1 VN ca vaccine strain replication and immunogenicity. Among the 9 amino acids that differed between the two H5 HA1 proteins, several HK03-specific residues enabled the VN04 ca virus to bind to both &#945;2,3SAL and &#945;2,6SAL receptors, but only the removal of the 158N glycosylation site together with an S227N change resulted in more efficient viral replication in the upper respiratory tract of ferrets and increased serum antibody response. However, the antibody response was HK03 strain-specific and did not significantly cross-neutralize VN04 virus. A second approach was taken to adapt the H5N1 VN04 ca virus in MDCK cells to select HA variants with larger plaque morphology. Although a number of large-plaque HA variants with amino acid changes in the HA receptor binding region were identified, none of these mutations affected virus receptor binding preference and immunogenicity. In addition, the known receptor binding site changes, Q226L and G228S, were introduced into the HA protein of the VN04 ca virus. Only in conjunction with the removal of the 158N glycosylation site did the virus replicate efficiently in the upper respiratory tract of ferrets and became more immunogenic, yet the response was also HK03-specific. Our studies indicate that the 158N glycosylation site and the receptor binding preference of the VN04 ca vaccine virus affect virus antigenicity and cause poor replication in the host resulting in a lower antibody response. In collaboration with scientists at Yale University, we analyzed the ability of a vaccine vector based on vesicular stomatitis virus (VSV) to induce a neutralizing antibody (nAb) response to avian influenza viruses in rhesus macaques. Animals vaccinated with vectors expressing either the A/Hong Kong/156/1997 or A/Vietnam/1203/2004 H5 hemagglutinin (HA) were able to generate robust nAb responses. The ability of the vectors to induce nAbs against homologous and heterologous avian influenza viruses after a single dose was dependent upon the HA antigen incorporated into the VSV vaccine. The vectors expressing the A/Vietnam/1203/2004 H5 hemagglutinin were superior to those expressing the A/Hong Kong/156/1997 HA at inducing cross-clade nAbs.